PROJECT SUMMARY/ABSTRACT Pathogenic variants in DNA damage repair (DDR) pathway genes are prevalent in a subset of men with metastatic castration-resistant prostate cancer (mCRPC) and occur in both the somatic and germline genomes. These abnormalities, primarily insertions and deletions resulting in protein truncations, occur in 10? 20% of patients with mCRPC. These variants also occur in lower-grade localized prostate cancers, although the prevalence of DDR mutations in these tumors and their impact on clinical outcome, survival, and drug sensitivity are largely unknown. In other projects of the proposed grant, specific mutations in DDR genes will be identified. The focus of Project 3 will be to prioritize and systematically evaluate these mutations, using a combination of functional assays, state-of-the-art organoid technology, CRISPR-mediated gene editing, and analysis of whole genome sequencing. In Aim 1 of Project 3, we will use multiple prostate cancer cell lines to determine whether specific DDR gene mutations are deleterious or benign variants. We will test the hypothesis that bona fide pathogenic DDR alleles are associated with a more aggressive tumor phenotype and will correlate with specific functional DNA repair defects and drug responses. In Aim 2, we will use gene editing to introduce DDR mutations into the endogenous genetic locus of multiple prostate cancer cell lines to provide a more physiologic assessment of the specific mutations. In Aim 3, we will analyze the whole-genome sequences from prostate cancers to identify precise mutational signatures which may result from loss-of- function mutations in specific DDR genes. Project 3 will have extensive collaborations with the investigators in Projects 1 and 2 and the Genomics Core as outlined in the attached research plan.